Role of IL-27-producing dendritic [corrected] cells in Th1-immunity polarization in Lewis rats

Lewis and Brown Norway rats are entirely different with respect to the polarization of their immune responses (Th1 and Th2, respectively). We found that naive Lewis rat splenocytes treated in vitro with heat-killed Mycobacterium tuberculosis (Mtb) upregulate the expression of both subunits of IL-27 (IL-27p28 and EBI3). Mtb treatment caused naive Lewis rat splenocytes to express 4.6-fold more IL-27p28 than Mtb-treated Brown Norway rat splenocytes 6h after the treatment. Although WSX-1, the IL-27 receptor, was not induced by Mtb treatment in splenocytes from either rat strain, Lewis rats expressed significantly higher levels of the IL-27 signal transducers T-bet and IL-12Rbeta2 than Brown Norway rats. Flow cytometric analysis of dendritic cells from bone marrow cells revealed Lewis rats had more IL-27p28-positive cells. Thus, early in the immune response, Lewis rats appear to produce higher levels of IL-27 than Brown Norway rats, resulting in polarization towards Th1-immunity.     

Soluble expression and purification of the functional interleukin-30 protein in Escherichia coli

Interleukin-30 (IL-30), or IL-27p28, is the α subunit of IL-27 constructed by Epstein–Barr virus-induced gene 3 (EBI3) and IL-27p28 binding via noncovalent bonds. IL-30 can be independently secreted and function independently of IL-27. Recent studies demonstrated IL-30 could concurrently antagonize T helper 1 (Th1) and Th17 responses and might have therapeutic implications for controlling autoimmune diseases. However, no reports have stated an efficient method to generate a relatively large quantity of IL-30. In this study, an Escherichia coli expression system for the rapid expression of the mouse IL-30 is developed. For the first time, IL-30 was expressed in a form of soluble fusion protein and purified using a method of simple affinity chromatography. In order to avoid the impact of minor codons on expressing eukaryotic protein in E. coli and to improve the expression quantity, the nucleotide sequence of IL-30 was optimized. The optimized gene sequence was then subcloned into the pET-44a(+) vector, which allowed expression of IL-30 with a fusion tag, NusA. The vector was transformed into E. coli and the expressed fusion protein, NusA-IL-30, was purified by Ni chromatography. Then the fusion tag was removed by cleavage with thrombin. The purity of purified IL-30 was identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as high-performance liquid chromatography (HPLC) and the purity was up to about 92%. The yield of IL-30 was 8.95 mg from 1 L of bacterial culture. Western blot confirmed the identity of the purified protein. The recombinant IL-30 showed its biological activity by inhibiting Th17 differentiating from naive CD4⁺ T cells. Therefore, this method of express and purifying IL-30 provides novel procedures to facilitate structural and functions studies of IL-30.     

The immunomodulating properties of human respiratory syncytial virus and immunostimulating complexes containing Quillaja saponin components QH-A, QH-C and ISCOPREP703

A successful vaccine against human RSV (HRSV) is likely to induce a Th1 or a balanced Th1/TH2 cytokine response. We tested a panel of HRSV immunostimulating complexes (ISCOMs) containing different Quillaja saponin fractions (QH-A, QH-C, and 703: a mixture of 70% QH-A and 30% QH-C) with different immunological properties for their capacity of inducing innate and acquired immune responses. The HRSV 703 ISCOMs induced the strongest innate and acquired immune responses, followed by RSV QH-C and QH-A ISCOMs. All three formulations induced various degrees of Th1 bias response with prominent production of IFN-gamma being 10-50 times higher than that of IL-4 and IL-5. The HRSV specific IgG isotype profile correlated with the predominant secretion of Th1 cytokines, with strong induction of IgG2a antibodies. The 703 ISCOMs induced the most pronounced Th1 profile followed by QH-C and QH-A ISCOMs. The high incorporation of F protein in these ISCOMs compared to G protein combined with the Th1 biased nature of ISCOM are likely to be the causes to promote a Th1 type of profile. The prospect to formulate an RSV ISCOM formulation with an optimal Th1/Th2 balance is in reach particularly in view of the versatile properties of the ISCOM concept.     

In vitro Th1 cytokine-independent Th2 suppressive effects of bifidobacteria

A comparison between 17 strains of lactic acid bacteria and 15 strains of bifidobacteria indicated that bifidobacteria induced significantly lower levels of interleukin-12 (IL-12) in murine splenic cells. The present study aims to evaluate the effect and mechanism of Bifidobacterium longum BB536, a probiotic strain, in suppressing antigen-induced Th2 immune response in vitro. BB536 suppressed immunoglobulin (Ig) E and IL-4 production by ovalbumin-sensitized splenic cells, but induction of Th1-inducing cytokine production, such as IL-12 and gamma interferon (IFN-gamma) tended to be lower compared with lactic acid bacteria. Neutralization with antibodies to IL-12, IFN-gamma, IL-10 and transforming growth factor beta indicated negative involvement of Th1-inducing cytokines and regulatory cytokines in the suppression of Th2 immune response by BB536, especially when treated at higher doses of BB536 (>10 microg cells/ml). Furthermore, BB536 induced the maturation of immature bone marrow-derived dendritic cells (BM-DCs), and suppressed antigen-induced IL-4 production mediated by BM-DCs. These results suggested that BB536 suppressed Th2 immune responses, partially independent of Th1-inducing cytokines and independent of regulatory cytokines, mediated by antigen-presenting cells such as dendritic cells.     

The role of the IL-33/ST2 axis in autoimmune disorders: Friend or foe?

Autoimmune diseases (ADs), which are common immune-mediated inflammatory syndromes, are characterized by an imbalance between T effector (Th)1/Th17 cells and T regulatory cells. Interleukin (IL)-33, a member of the IL-1 family, induces inflammatory disease development by mediating type 2 immune responses. Recently, IL-33/ST2 axis was reported to induce autoimmunity involving Th1 and Th17 cells. In this review, we focus on the expression, regulation and function of IL-33/ST2 pathway in the context of autoimmune disorders. We discuss the clinical potential of this signaling pathway in predicting disease activity and severity and offer possible future therapeutic alternatives.     

原发性肝癌患者Th1/Th2 亚群研究*

目的:探讨原发性肝癌患者血清中淋巴细胞亚群中Th1/Th2的变化,为分析肝癌的发生发展状症和临床治疗提供免疫学指标。方法:应用放射免疫分析及酶联免疫分析法(ELISA),测定46例肝癌患者,及43例正常对照组进行比较。以IL-2、INF-γ和TNF-α水平代表Th1型细胞因子,以IL-4,IL-6、IL-8、IL-10的水平代表Th2型细胞因子。结果:肝癌患者IL-2、TNF-γ、IL-6的水平明显低正常对照组,P<0.01。IL-4、IL-8、IL-10、TNF-α的水平明显高于正常对照组,P<0.01。结论:肝癌患者体内存在Th1/Th2细胞因子失衡,其中Th1亚群功能抑制,Th2亚群功能亢进,其与肿瘤在宿主体内生长密切相关。通过纠正这些免疫失调将成为肝癌治疗的重要手段。     

原发性肝癌患者Th_1/Th_2亚群研究

目的：探讨原发性肝癌患者血清中淋巴细胞亚群中Th1/Th2的变化,为分析肝癌的发生发展状症和临床治疗提供免疫学指标。方法：应用放射免疫分析及酶联免疫分析法（ELISA）,测定46例肝癌患者,及43例正常对照组进行比较。以IL-2、INF-γ和TNF-α水平代表Th1型细胞因子,以IL-4,IL-6、IL-8、IL-10的水平代表Th2型细胞因子。结果：肝癌患者IL-2、TNF-γ、IL-6的水平明显低正常对照组,P〈0.01。IL-4、IL-8、IL-10、TNF-α的水平明显高于正常对照组,P〈0.01。结论：肝癌患者体内存在Th1/Th2细胞因子失衡,其中Th1亚群功能抑制,Th2亚群功能亢进,其与肿瘤在宿主体内生长密切相关。通过纠正这些免疫失调将成为肝癌治疗的重要手段。     

IL-4 is required to prevent filarial nematode development in resistant but not susceptible strains of mice

The murine Litomosoides sigmodontis model of filarial infection provides the opportunity to elucidate the immunological mechanisms that determine whether these nematode parasites can establish a successful infection or are rejected by the mammalian host. BALB/c mice are fully susceptible to L. sigmodontis infection and can develop patent infection, with the microfilarial stage circulating in the bloodstream. In contrast, mice on the C57BL background are largely resistant to the infection and never produce a patent infection. In this study, we used IL-4 deficient mice on the C57BL/6 background to address the role of IL-4 in the development of L. sigmodontis parasites in a resistant host. Two months after infection, adult worm recovery and the percentage of microfilaraemic mice in infected IL-4 deficient mice were comparable with those of the susceptible BALB/c mice while, as expected, healthy adults were not recovered from wild type C57BL/6 mice. The cytokine and antibody responses reveal that despite similar parasitology the two susceptible strains (BALB/c and IL-4 deficient C57BL/6) have markedly different immune responses: wild type BALB/c mice exhibit a strong Th2 immune response and the IL-4 deficient C57BL/6 mice exhibit a Th1 response. We also excluded a role for antibodies in resistance through infection of B-cell deficient C57BL/6 mice. Our data suggest that the mechanisms that determine parasite clearance in a resistant/non-permissive host are Th2 dependent but that in a susceptible/permissive host, the parasite can develop in the face of a Th2 dominated response.     

Understanding the pro- and anti-inflammatory properties of IL-27

The recent identification of IL-27 (IL-27p28/EBV-induced gene 3) and IL-27R (WSX-1/gp130) has provided new insights for the biology of IL-6/IL-12 family cytokines. Initial studies indicated that IL-27 can directly regulate T cell functions and suggested an important role for it in promoting Th1 type responses. However, subsequent studies have revealed that IL-27R signaling influences a variety of immune cell types and can inhibit either Th1 or Th2 type responses. Though elucidation of the Jak/STAT signaling pathways activated by IL-27R ligation has unveiled some of the molecular mechanisms used by IL-27 to promote inflammation, little is known about the anti-inflammatory activities of this cytokine. Thus, the aim of this review is to discuss the pleotropic nature of the IL-27/IL-27R interaction and attempt to reconcile the pro- and anti-inflammatory properties of this immunomodulator.     

The attenuation of Th1 and Th17 responses via autophagy protects against methicillin-resistant Staphylococcus aureus-induced sepsis

Whether autophagy affects methicillin-resistant Staphylococcus aureus (MRSA)-induced sepsis and the associated mechanisms are largely unknown. This study investigated the role of autophagy in MRSA-induced sepsis. The levels of microtubule-associated protein light chain 3 (LC3)-II/I, Beclin-1 and p62 after USA300 infection were examined by Western blotting and immunohistochemical staining. Bacterial burden analysis, hematoxylin-eosin staining, and Kaplan–Meier analysis were performed to evaluate the effect of autophagy on MRSA-induced sepsis. IFN-γ and IL-17 were analyzed by ELISA, and CD4⁺ T cell differentiation was assessed by flow cytometry. Our results showed that LC3-II/I and Beclin-1 were increased, while p62 was decreased after infection. Survival rates were decreased in the LC3B^−/− and Beclin-1^+/− groups, accompanied by worsened organ injuries and increased IFN-γ and IL-17 levels, whereas rapamycin alleviated organ damage, decreased IFN-γ and IL-17 levels, and improved the survival rate. However, there was no significant difference in bacterial burden. Flow cytometric analysis showed that rapamycin treatment decreased the frequencies of Th1 and Th17 cells, whereas these cells were upregulated in the LC3B^−/− and Beclin-1^+/− groups. Therefore, autophagy plays a protective role in MRSA-induced sepsis, which may be partly associated with the alleviation of organ injuries via the downregulation of Th1 and Th17 responses. These results provide a nonantibiotic treatment strategy for sepsis.     

IL-27 suppresses Th2 cell development and Th2 cytokines production from polarized Th2 cells: a novel therapeutic way for Th2-mediated allergic inflammation

IL-27 up-regulates Th1 but down-regulates Th2 responses. However, its molecular mechanism and regulatory effects on polarized Th2 cells remain unclear. In this study, we have revealed that IL-27 inhibits Th2 cell development as well as Th2 cytokines production from already polarized Th2 cells by down-regulation of GATA-3 and up-regulation of T-bet expression simultaneously. In vivo daily IL-27 treatment for 1 wk after Leishmania major infection protects BALB/c mice from footpad swelling by diminishing parasite burden via reciprocal regulation of Th1 and Th2 responses. Furthermore, IL-27 stimulation causes marked reduction in the capacity of host mouse to mount a Th2 response against Strongyloides venezuelensis infection. Thus, IL-27-treated mice failed to develop intestinal mastocytosis after S. venezuelensis infection and exhibited a marked delay in parasite expulsion. Finally, intranasal administration of IL-27 inhibits OVA-induced airway hyperresponsiveness and inflammation in OVA-sensitized animals. Thus, IL-27 could provide us with a novel therapeutic way for treating Th2-associated diseases such as bronchial asthma.     

Contribution of IL-12R mediated feedback loop to Th1 cell differentiation

T helper 1 (Th1) cell fate is induced by overlapping signaling pathways, whose kinetic principles and regulatory motifs are largely unknown. We identified a simple positive feedback loop in the STAT4 signaling pathway, whereby activation by IL-12 leads to the increased expression in IL-12 receptor. A computational analysis shows that this feedback loop synergizes with the one mediated by the IFN-gamma secreted by differentiating cells, when the induction of Th1 cell fate is weak. Positive feedback loops are often utilized to enhance phenotypic differentiation. This effect was confirmed by experiments showing that stochastic fluctuations in the expression of IL-12 receptor gene were amplified, leading to two discrete levels of expression in a cell population.     

Dendritic cells post-maturation are reprogrammed with heightened IFN-gamma and IL-10

Mature dendritic cells (mDCs) undergo "exhaustion" in producing cytokines. Nevertheless, whether this "exhaustion" of mDCs is selective to certain cytokines, or whether mDCs have specific cytokine-producing profiles has yet to be defined. Herein, we investigated the cytokine production in vitro by immature DCs (iDCs) and LPS-induced mDCs. Compared to iDCs, mDCs produced comparable levels of IL-6 and TNF-alpha. Strikingly, mDCs produced significantly higher IFN-gamma and IL-10. IL-12 production of mDCs was suppressed. Kinetic studies of the responses of iDCs and mDCs to LPS or CD40L showed that mDCs acquired progressively heightened activity in producing IFN-gamma and IL-10. TNF-alpha-, IL-6-producing capability of mDCs was maintained. Nevertheless, IL-12 production by mDCs was not recovered at any time point. Mature DCs were potent in priming both Th1 and Th2 cells. In conclusion, upon maturation, DCs are reprogrammed with a distinct cytokine-secreting profile, which may play an important role in regulating T cell functions.     

Altered T helper responses in CD40 and interleukin-12 deficient mice reveal a critical role for Th1 responses in eliminating the helminth parasite Taenia crassiceps

A key feature of helminth infections is the induction of strong Th2-biased immune responses in their hosts. We have previously found that Th2-like responses mediate susceptibility to the helminth parasite Taenia crassiceps, probably by inhibiting Th1 responses required for the development of protective immunity against this parasite. Here we show that mice lacking interleukin-12p35 (IL-12p35-/-) following T. crassiceps infection, failed to mount a Th1 response, but developed a strong Th2-type response, produced higher levels of IgG1, IgE, interleukin-4, interleukin-5 as well as interleukin-13 than wild-type mice, and became highly susceptible to the larval stage of this cestode. In contrast, similarly-infected CD40 deficient BALB/c mice (CD40-/-) displayed impairment of both Th1 and Th2-type responses associated with low levels of interferon-gamma as well as IgE, interleukin-4, interleukin-5 and interleukin-13, but efficiently controlled T. crassiceps infection. Together, these findings suggest a detrimental role for Th2-biased responses during the larval stage of T. crassiceps infection. Furthermore, they also suggest a pivotal role for CD40 in developing Th2-type responses.     

In vivo study: Th1–Th17 reduction in pristane-induced systemic lupus erythematosus mice after treatment with tolerogenic Lactobacillus probiotics

Uncontrolled inflammation in systemic lupus erythematosus (SLE) could cause dysfunction in multiple organs. T helper 17 (Th17) cells are a main branch of inflammatory responses in the pathogenesis of SLE, and by producing interleukin 17 (IL-17), represent a major functional tool in the progression of inflammation. Animal models provide a special field for better studies of the pathogenesis of diseases. Tolergenic probiotics could decrease inflammation in autoimmune diseases by modulating the immune system and maintaining homeostasis. The aim of this project was to evaluate the effects of Lactobacillus rhamnosus and Lactobacillus delbrueckii on Th17 cells and their related mediators in a pristane-induced BALB/c mice model of SLE. The mice were divided into pretreatment groups, which received probiotics or prednisolone at Day 0, and treatment groups, which received probiotics and prednisolone 2 months after injection. The presence of antinuclear antibody (ANA), anti-double-stranded DNA (anti-dsDNA), and anti-ribonucleoprotein (anti-RNP) and lipogranuloma was evaluated; also, the population of Th1–Th17 cells as well as interferon γ (IFN-γ), IL-17, and IL-10 levels, and the expression of RAR-related orphan related receptor gamma (RORγt) and IL-17 were determined. We observed that probiotics and prednisolone could delay SLE in pretreatment and treatment mice groups, with a reduction in ANA, anti-dsDNA, anti-RNP, and mass of lipogranuloma. Probiotics and prednisolone decreased the population of Th1–Th17 cells and reduced IFN-γ and IL-17 as inflammatory cytokines in the pretreatment and treatment groups in comparison with SLE-induced mice. Our results indicated that, due to their anti-inflammatory properties and reduction of Th17, Th1, and cytotoxic T lymphocyte (CTL) cells, the use of these probiotics could probably represent a new tool for the better management of SLE.     

Contrasting effects of acute and chronic gastro-intestinal helminth infections on a heterologous immune response in a transgenic adoptive transfer model

We have previously found that co-immunisation with ovalbumin (OVA) and the body fluid of the helminth Ascaris suum inhibited an OVA-specific delayed type hypersensitivity (DTH) response by reducing OVA-specific CD4+ T lymphocyte proliferation via an IL-4 independent mechanism. In the present study, we determined whether parasite infections themselves could induce similar changes to peripheral immunisation by examining the modulation of OVA-specific immune responses during acute and chronic helminth infections. Surprisingly, an acute infection with Trichinella spiralis, but not a chronic infection with Heligmosomoides polygyrus, inhibited the OVA-specific DTH reaction. Correspondingly, the T helper 1 (Th1) OVA-specific response was decreased in mice infected with T. spiralis, but not with H. polygyrus. Inhibition of the Th1 response may be a result of a shift in the Th1/Th2 balance as although both H. polygyrus and T. spiralis infected mice induced a Th2 OVA-specific response, that exhibited by T. spiralis was more potent. Furthermore, although IL-10 secretion upon OVA restimulation was similarly increased by both infections, production of this immunoregulatory cytokine may play a role in the suppression of immune responses observed with T. spiralis infection depending on the context of its release. Interestingly, analysis of the OVA-specific T lymphocyte division by carboxyfluorescein diacetate succinimidyl ester (CFSE) staining revealed that gastro-intestinal infection with the acute helminth T. spiralis, but not with chronic H. polygyrus, inhibited the systemic immune response by significantly inhibiting the antigen-specific T cell proliferation during the primary response, a mechanism similar to that observed when A. suum parasite extracts were directly mixed with the OVA during immunisation in our previous studies.